Seppro was specifically designed for immunoaffinity partitioning of proteins by removing the 14 highly abundant proteins (HAP) from various biological samples, which has proven to be useful for analysis of low-abundant proteins (LAP) and biomarker discovery. (Top)

4. What types of projects are best fit for Seppro?

By eliminating the great majority of interfering proteins, Seppro has been demonstrated to enhance 2-Dimensional Gel Electrophoresis (2DE) and Mass Spectrometry (MS) to detect those LAP previously inaccessible or masked by the HAP. (Top)

5. What are the immediate assays that can be used after processing samples using Seppro?

6. What is Sigma's Seppro line of immunoaffinity separation reagents for proteomics sample processing, and how do they compare to the competition?

We think that Seppro is the ultimate application for IgY antibodies, playing directly to their technical strengths. Sigma has a patented technology to covalently link in an oriented fashion “affi-pure” IgYs directed against abundant plasma/serum proteins on the surface of microbeads specifically designed for IP applications. While antigen binding and specificity is quantitatively higher than the goat IgG-based solutions offered by our competitors, Seppro is qualitatively better for separating non-human mammalian antigens such as albumin, fibrinogen and transferrin from rat, mouse, or dog sera. This enables highly efficient toxicoproteomics & biomarker discovery, and confirmation of animal disease models. (Top)

7. How much of the abundant plasma proteins (HAP) can Seppro remove?

We estimate based on relative protein abundance in serum or plasma that Sigma's IgY-14 product removes more than 95% of the abundant proteins, enhancing biomarker discovery more than 20-fold compared with un-fractionated plasma or serum. (Top)

8. After I discover putative biomarkers using IgY12/ IgY-14 and proteomics, how can Sigma help?

We produce gene-specific IgY antibodies directed just against the putative biomarker protein. These can be used for Western Blots, IP, IHC, cell sorting and other immunoassays. (Top)

9. What are the storage requirements for Seppro products?

Immunoaffinity separation columns should be stored at 2-8°C. For long-term storage to prevent microbial growth, we recommend addition of 0.02% sodium azide to the Dilution Buffer in the column only. All other kit components (buffers, spin columns, collection tubes, etc.) can be stored at room temperature.(Top)

10. Is there any sample pretreatment required (filtering, desalting etc.)?

After dilution in Dilution Buffer, serum and plasma samples should be prefiltered through the supplied 0.45um spin filters to remove particles and other debris (e.g., fibrin clots) that could foul the LC column. (Top)

11. Can I use this for any species of plasma or serum (human, dog, monkey, rat, etc.)?

The mixture of antibody-coupled microbeads in IgY-14 was optimized for human and primate sera or plasma. While many of these antibodies are known to cross-react very well with orthologous proteins from other mammalian species, IgY-14 is not recommended for non-human/primate samples due to different natural abundances and ratios. 13. Can I recover the albumin/abundant proteins?

It is very simple to recover and subsequently analyze the abundant proteins captured by IgY Microbead columns. Simply collect the second, eluted peak containing >90% of the plasma proteins and add 1/10 volume Neutralization Buffer. Then concentrate by ultrafiltration or precipitate with ethanol, TCA or acetone. (Top)

14. What other proteins will be removed?

No currently available method is more specific than immunoaffinity capture. Only the specific proteins indicated will be removed from the plasma or serum samples. (Top)

15. What are the physical properties of IgY-Microbeads?

The solid support used in Seppro IgY products is hydrophilic, charge-free, high-capacity, highly cross-linked, rigid, copolymeric and porous, resulting in minimal nonspecific interactions with the sample. It’s a medium pressure liquid chromatography resin, capable of withstanding pressures up to 100psi (6 bar). (Top)

16. How can I tell when the column is exhausted?

The columns are rated for 100 cycles. We recommend discarding the columns after this number of cycles have been run. Empirical evidence on maintenance of continued antibody specificity and capacity can be obtained by chromatogram shape, 1D SDS-PAGE or mass spec analyses. Significant changes in expected column behavior could be due to fouling, and can generally be prevented by sterile filtration through 0.45um spin filters after dilution but prior to injection into the LC column. (Top)

17. Can it be regenerated?

Each cycle involves the following 5 steps using three buffers: binding, washing, elution, neutralization and re-equilibration. Typically, the researcher collects the flow-through/partitioned peak(s) and the bound/eluted peak. As mentioned, we recommend discarding the column after 100 cycles. (Top)

18. How long is the run time?

It depends on the LC equipment used with the column, but at normal flow rates a typical LC2 run takes 48 min; a typical LC10 run takes 70 min. (Top)

19. Can I make the buffers myself?

Absolutely. The Dilution and Neutralization Buffers are standard. It is particularly important to properly calibrate one’s pH meter before attempting to make Stripping Buffer, since pH 2.50 is crucial for removing bound proteins while avoiding acid degeneration of the IgY antibodies bound to the column. (Top)

20. Can the columns be used with any LC system?

Yes. The standard 1/4-28 fittings supplied with the LC columns can be used with appropriate adapters to FPLC (M6 fittings) or HPLC (10-32 fittings). We recommend using Tefzel, Teflon or PEEK materials, with inlet and outlet tubing of 1/16” OD. Ensure that all organic buffers are first removed; the system must be equilibrated with ProteomeLab kit buffers before use. (Top)

21. Can I use this for CSF, urine or other biofluids?

Yes. As long as human samples are sterile-filtered through the 0.45um spin filter to remove particulate material before injection into the column, and capacities are optimized, these samples should fractionate well. (Top)

No. There are no detergents, surfactants, urea, other chaotropic agents or preservatives in any of the buffers. The shipped IgY Microbead resins only have 0.02% sodium azide added as an anti-microbial preservative. (Top)

24. Do I need to exchange the buffer from the flow-through fraction before analysis?

The flow-through fraction is in TBS (Tris-Buffered Saline). Protein in this fraction (and the bound/eluted fraction) can be concentrated through a spin ultrafiltration membrane, or precipitated with ethanol, acetone or TCA. (Top)

25. What is the carry-over from sample to sample?

After proper recycling of the column, no protein was detected by mass spec analysis. An optional stripping cycle can be added to analyze any residual bound protein. (Top)

About 5-10% of total primate plasma protein is recovered in the flow-through/partitioned fraction using the IgY-14 column. About 15% of rodent plasma protein is recovered in the flow-through fraction using the IgY Rat column. (Top)

28. Why do I need to transfer the IgY Microbeads into a fresh spin column after 25 cycles?

While the IgY-Microbead resin is durable for 100 cycles under proper conditions, the spin column frit flow rates sometimes drop after 30 uses. For this reason, we recommend transferring the 50% slurry with a 1 mL disposable pipette tip into a fresh spin column after 25 cycles. By repeating this transfer three times (after cycles 25, 50 & 75), the spin column resin can be used 100 times. We feel that this simple mechanical transfer is more reliable than including chaotropic agents, such as urea, in the stripping buffer.(Top)

29. What is the total yield (yield per use, uses per spin column, and total yield per kit) of partitioned proteins using the IgY-14 and HSA spin columns?

The serum/plasma loading capacity of the IgY12 spin column is 10 µL. Assuming a total serum/plasma protein range of 70-80 mg/mL, one can load 700-800 µg protein on the IgY12 spin column. About 90% of the total protein is bound to the IgY-14 column, yielding an eluted quantity of 720 µg. About 10% of the total protein is present in the flow-through fraction, yielding a quantity of about 80 µg with the 12 most abundant proteins removed. Since each spin column can be used 100 times, the total yield is ~8 mg of partitioned protein. Since a kit contains 2 spin columns, each IgY12 Spin Column kit should yield a total of about 16 mg of IgY12-partitioned protein. (Top)

30. I’m not just interested in depleting serum of abundant proteins, but also in what physiologically binds to serum albumin. Is Seppro good for this application?

The serum protein complex bound to an anti-HSA IgY is even cleaner than to an anti-HSA monoclonal IgG column. This must be due to less background binding to the Fc portion of IgY compared to IgG, enabling effective Albuminomics™. Virtually every lab that has tested Sigma's anti-HSA IgY has been very impressed with its exquisite specificity. (Top)